Fig. 2

Identification of efficient NMN⁺ production pathways in vivo using a growth-based screening platform. A growth-base screening platform was used to identify pathways which efficiently generated NMN⁺ in vivo. E. coli strain 72c, which contains a temperature sensitive allele of nadD (ts nadD), which exhibits a conditionally lethal phenotype when cultured at 42 °C because the native NAD⁺ biosynthesis is disrupted. Therefore, the cell must rely on intracellular NMN⁺ to restore NAD⁺ formation and growth. a Direct feeding of NMN⁺ into the growth medium with the overexpression of an NMN⁺ transporter, PnuC* from S. enterica, restored growth to levels near the wild type control, indicating the platform is effective for NMN⁺ production screening. b Introducing F. tularensis NadE* also restored growth with the supplementation of nicotinamide. c, d Overexpression of E. coli ‘s native nicotinamide riboside (NR) transporter PnuC, paired with S. enterica NadR (c) or S. cerevisiae Nrk1 (d) while feeding NR failed to efficiently restore growth. Screening was performed in a deep-well 96-well plate containing 1 mL of LB medium supplemented with 2 g/L d-glucose and 200 μM of NMN⁺ precursors, if applicable